Nutrient Metabolism - Enzyme Regulation

Regulation by enzyme quantity -

-Balance between rate of synthesis vs. rate of degradation. -Substrate can induce enzyme synthesis via DNA transcription (slow process).

Regulation by compartmentation -

-FA synthesis (cytosol) vs. FA oxidation (mitochondria). -Separation by metabolic state (glucose degradation, i.e., glycolysis vs. glucose synthesis, i.e., gluconeogenesis).

Mechanisms of Enzyme Regulation -

1. Compartmentation. 2. Substrate availability: Vmax and Km. 3. Enzyme quantity. 4. Regulation by product inhibition. 5. Allosteric regulation. 6. Regulation by covalent modification.

• Allosteric enzymes share several characteristics: • The effector binds the allosteric enzyme into a regulatory site other than the ___ site. • Allosteric proteins are oligomeric proteins (i.e., they have ___ structure) composed of two or more subunits. This provides multiple ligand binding sites, for ___. The binding of the effector produces a ___ change that is going to be transmitted from one subunit to the other, producing a change in enzyme activity. • The effector binds non-covalently, i.e., the binding is ___. Since the binding of the effector is at a site different from the active site, there is no requirement for ___ between substrate and effector. • Allosteric enzymes show a ___ curve when the initial reaction velocity is plotted against substrate concentration. • Since allosteric enzymes do not follow Michaelis- Menten kinetics, we do not use Km; • We use ___, which is also defined as the concentration of substrate where the velocity of the reaction is half of the maximal. • Allosteric enzymes exhibit positive ___: Binding of first substrate to enzyme changes conformation increasing the affinity of the next substrate to bind other subunits of enzyme/protein. Example O2 binding to hemoglobin.

Active (catalytic); quaternary; substrates and effectors; conformational; reversible; structural similarity; sigmoidal; K0.5; cooperativity.

How do enzymes work in glycolysis?

At each step, an enzyme controls the conversion rate. The product of one reaction is the substrate of the subsequent reaction.

The flux of substrates is analogous to _______ traveling on a highway, with the rate limiting step as a barrier or a traffic-light in the highway.

Cars.

The mechanism by which many of these regulatory strategies work lies at the level of -

Changing the conformational state of the enzyme.

Enzymes can also be regulated through -

Covalent modifications and proteolysis.

Drugs are interested in what allosteric effector mechanism?

Drugs interested in inhibition (Km increase and affinity decrease). DPP4 is inhibitor for diabetes. Januvia helps patients via enzyme inhib.; statins are also inhibitors. - AMPK activators. Some drugs also interested in activation.

Fed vs. Fasted state Km and affinity -

Fed state - glucokinase in liver; high Km (10 mM glucose), low affinity. Fasted state hexokinase; low Km (0.1 mM glucose), high affinity

The controls our cells use to turn-on or turn-off different biological systems often work via regulation of -

Enzyme activity.

More ___ = more products; can synth enzyme from ___ but takes a long time (goes to DNA).

Enzymes; substrate.

Another type of allosteric inhibition is -

Feedback inhibition is the ability of the product in the metabolic pathway to inhibit the activity of the first enzyme in the pathway, thereby ensuring that the functioning of the pathway is sensitive to the intracellular concentration of the product. Example: Inhibition of threonine deaminase by isoleucine, the end product of the pathway. *Enzymes should not be considered as on/off switches, but as a thermostat.

Rate limiting enzyme checks each car so can go to next step (picture) - traps and can only move ___. Some proteins can ___ rate-limiting step - like cars. ___ is destination for car.

Forward; bypass; product.

Virtually all of our metabolic pathways are regulated in order to maintain -

Homeostasis.

sensitive means of communication.

Interconnected.

The rate-limiting step is an ___________ reaction of the pathway.

Irreversible.

Irreversible covalent modification refers to _______________, which is the one-time, irreversible removal of a portion of a polypeptide chain by an appropriate proteolytic (protein-degrading) enzyme. • Many enzymes are produced in an inactive precursors form, called _____________. In order to become an enzyme, the proenzyme must be selectively cleaved. • Proteolytical cleavage results in an irreversible conformational change that exposes the active site. • Example: Proteolytic enzymes of the pancreas (trypsin, chymotrypsin, carboxipeptidase) are synthesized and secreted in an inactive form into the duodenum. Why??

Limited proteolysis; Proenzymes or zymogens; as a zymogen called trypsinogen. When trypsinogen reaches the duodenum, it is activated by the removal of a hexapeptide from its N-terminus by the action of enterokinase, a membrane-bound protease produced by the duodenal cells. The active trypsin then actiates other zymogens by specific proteolytic cleavages.

Multi-enzyme pathways can be ________ or _______, and they are all _____________. Since some metabolites are shared among pathways, the fate of a certain metabolite affects the other pathways.

Linear or branched; interconnected.

The flux of ___ involves many enzymes, but the active control is achieved by the regulation of a few of them.

Metabolites.

In our cells, groups of enzymes work together in specific metabolic pathways consisting of several steps. They are called -

Multi-enzyme pathways.

Why secrete proenzymes in inactive forms?

Not in active until needed, we can activate to use it whenever we need it. o Pancreas secretes pancreatic kinase, +2 all 3 are inactive. We secrete enterokinase o Cystic fibrosis - ducts are blocked. Hard time digesting Proteins, Carbs, Fats.

Describe an idealized cell in steady state -

Nutrients in; large molecules and small molecules broken down/formed in relation to fasted/fed state; Phosphorylation & Dephosphorylation occur; wastes out.

Reversible covalent modifications -

Phosphorylation: - Protein kinase catalyzes transfer of phosphate group from ATP to hydroxyl group of specific serine, threonine, or tyrosine. - Protein phosphatase catalyzes hydrolysis of phosphate group.

Covalent bonding can control enzyme activity. E means dephosphorylation; EP is phosphorylation. Varies which activates enzyme. Phosphorylation is just a ___ modification. For some, will activate or deactivate enzyme.

Post-translational (doesn't change DNA).

Lastly, enzymes can be regulated at the level of -

Protein synthesis and degradation.

The slowest step in the pathway is the _________________. It is going to determine the flow through the whole pathway; it's like a quality check.

Rate-limiting step.

By increasing or reducing the rate of a ___________step, the entire pathway is going to be controlled.

Rate-limiting.

Most ____ enzymes are controlled through active regulatory mechanisms that change the ___ in a way that affects the ___.

Rate-limiting; conformation of the enzyme; catalytic site.

Rate-limiting steps are targets of regulation and targets of intervention by drugs. Ex.: ___________control the rate- limiting step in cholesterol synthesis.

Statin drugs.

There are several types of mechanisms to regulate enzymes. Metabolic pathways can be regulated by -

Substrate availability, product and feedback inhibition.

Allosteric effectors can be -

Substrates, products, intermediates. Either Homotrophic (substrates themselves are effectors) or Heterotrophic (effector is different than substrate). Activators or inhibitors: Activators increase enzyme activity & shift curve to the left (Km decrease, affinity increase). Inhibitors decrease enzyme activity & shift curve to the right (Km increase, affinity decrease).

Regulation by covalent modification -

The activity of an enzyme is regulated by the addition or removal of a specific chemical group via covalent bonding. The effect is immediate and can be either reversible or irreversible - Addition of phosphate groups, methyl groups, acetyl groups, or derivatives of nucleotides (eg., adenylation, uridylation)

Allosteric enzymes can be positively or negatively regulated by -

The binding of allosteric effector molecules.

In many cases, once the first reaction has proceeded -

The flow can't go backwards. It is committed to proceed due to energy considerations.

Regulation by Allosteric Inhibition -

The word allosteric is derived from the Greek word Allos (other) + Steros (site/shape). • Allosteric enzymes change their conformation (shape) in response to the binding of molecules called effectors (or modulators).

Regulation by Product Inhibition -

When the concentration of product from an enzyme-catalyzed reaction builds up, the product may inhibit the activity of the enzyme. This inhibition is usually carried out by the immediate product of a reaction (B in the figure), but may be a product downstream in the pathway (C or D in the figure). • Products display structural similarity to the substrate. • Products can bind reversibly to the active site. • The response of the enzyme is immediate. • Example: Product inhibition of hexokinase by glucose-6-phosphate. In picture, b is inhibiting enzyme 1 so a doesn't make so much more b.

The Why, When, Where, and How of enzyme regulation -

Why: To maintain homeostasis because conditions change constantly. When: All the time. Where: In every cell and tissue. How: Different mechanisms.